| Objective:1.To explore the effect and possible mechanism of subchronic PAH mixture exposure on neurobehavioral changes in male SD rats.2.Differentiallly expressed miRNAs in hippocampus were selected by gene microarray.The bioinformatics analysis of the related miRNAs was performed to provide more clues at the molecular level to further explore the mechanism of PAH neurotoxicity.Methods:1.Forty healthy male SPF Sprague-Dawley rats were randomly divided into 4 groups,with ten rats in each group.They were control group,low,middle and high exposure group(rats received intraperitioneal injection of vegetable oil,PAH mixture 3.2,8.0,20.0 mg/kg body weight once in two days for 60 days,respectively).At the end of exposure,Morris water maze test was performed to detect spatial learning and memory function of rats.Open field test was adopted to determine locomotor activity and exploratory behaviors of rats.2.After behavior experiments were completed,hippocampus tissues were obtained from brain of decapitated rats.The Reactive oxygen species(ROS)content was determined by the Chemiluminescence method,the Malondialdehyde(MDA)content was determined by thiobarbituric acid method,and the Superoxide dismutase activity(SOD)was determined by the water-soluble nitroblue tetrazolium salt method.The monoamine neurotransmitters(Noradrenaline,Epinephrine,Dopamine,5-Hydroxytryptamine)levels were measured with enzyme-linked immunosorbent assays.The Acetylcholine level was measured by the microplate method.The Acetylcholinesterase activity was determined by the colorimetry.The Glutamic acid level was measured by the UV colorimetry.3.RNA samples were isolated from the hippocampus homogenates.Differentiallly expressed miRNAs in hippocampus were selected by gene microarray.The predicted target genes of the differentially expressed miRNAs were obtained using Targetscan database.GO and KEGG pathway analysis were performed to explore the functional annotation of candidate target genes.4.Statistical analyses were carried out using SAS software for windows,ver.9.4 and Graphpad Prism 7.0.The statistical level was ?=0.05.Results:1.Body weight change in rats after PAH exposure: After 56 days of PAH exposure,body weight of high level group was lower than the control group(P<0.05).2.Morris water maze test: Compared with the control group,the time spent in the target quadrant was shorter in the high,middle,and low level exposure group(P<0.05);the rest time in the target quadrant was longer than low and middle level exposure group(P<0.05).3.Open field test: Standing time was significantly different between the groups(P<0.05).Standing time in high level group was lower than that in the control and low level group(P=0.017;P=0.010).4.Oxidative stress response in rats: ROS content was significantly different between the groups(P<0.05),ROS content in middle level group was higher than that of the control group(P=0.007).5.Monoamine neurotransmitters in rats: There was a statistically significant difference in DA content between the groups(P<0.01).The content in low,middle and high level group was higher than that in the control group(P=0.016;P=0.002;P=0.001).6.Cholinergic neurotransmitters in rats: There was no significant difference in Ach content and AchE activityty between the groups(F=0.466,P=0.709;F=0.105,P=0.956)7.Amino acid neurotransmitter in rats: There was a statistically significant difference in Glu level between the groups(P<0.05).Glu level in low and high level group was lower than that in the control group(P<0.05).8.PAH exposure leads to changes of miRNA expression profiles in rat hippocampus.A total of 29 miRNAs were identified,1 miRNA was more than 1.5 times up-regulated,and 28 miRNAs were more than 1.5 times down-regulated.9.Target gene prediction and bioinformatics analysis:We selected three miRNAs(miR-211-3p,miR-667-3p and miR-3575-3p)with down-regulated expression in three comparison groups for target gene prediction revealed that 315 target genes were predicted from Targetscan database.GO enrichment analysis shows target genes were mainly enriched in biological process,molecular function,cellular component,protein binding,intracellular sodium activated potassium channel activity,glycosphingolipid biosynthesis-lacto and neolacto series,RNA transport,and so on.Likewise,KEGG pathway analysis showed many important pathways,such as Arrhythmogenic right ventricular cardiomypathy pathway,glycosphingolipid biosynthesis-lacto and neolacto pathway,RNA transport pathway,and so on.Conclusions:1.PAH exposure may cause growth retardation,learning and memory abilities decline and anxiety-like behavior in male SD rats.2.Impaired neurobehavioral changes in rats induced by PAHs may be related to oxidative stress,changes in neurotransmitters content,and changes in miRNA expression. |
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